experimental control and modification of larval development in the sea urchin in relation to the...

EXPERIMENTAL CONTROL AND MODIFICATION OF LARVAL DEVELOPMENT IN THE SEA URCHIN

IN RELATION TO THE AXIAL GRADIENTS

C. M. CHILD Hull Zoological Laboratory, University of Chicago

EIGHT PLATES

The literature of experimental teratogeny in embryonic and larval development is extensive and a great variety of external factors has been employed in experimentation along this line. While the results have often been of great interest, no general basis for interpretation has been reached, particularly as regards the relation between teratological form and the action of experimental factors to which the developing organism is sub- jected as a whole, and not by local application. It is a familiar fact that even such factors affect different regions or parts dif- ferently or to a different degree, and while such differences indicate the existence of local differences of some sort, there is, in general, a very evident lack of specificity in the action of different external factors on the course of organic development. Simi- lar teratological forms may often be produced by many different agents and conditions.

The present paper constitutes one step in the attempt to cor- relate certain types of teratological development with dynamic conditions which are characteristic and fundamental features of the normal organism. It establishes a basis for the control, modification and prediction of the course of development in the sea urchin, and the facts already a t hand indicate very clearly that certain types of teratological development, as well as certain characteristic features of normal development, can readily be interpreted on the same basis. More specifically this paper is a demonstration of the effectiveness of the axial metabolic gradients as dynamic factors in the development of the sea

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JOURNAL OF MOXPHOLOOY, VOL. 28, NO. 1

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urchin. It shows how, and to what extent, it is possible to control and modify development by means of the differential action of external factors on different regions of these gradients.

THE NATURE AND PURPOSE OF THE EXPERIMENTS

The existence of an axial metabolic gradient, at least in the major or polar axis with the apical region as the region of highest rate of reaction, or, perhaps, as possessing the capacity for the highest rate of reaction has been demonstrated in many species of animals and plants (Child, '13 b, '14 a, '15 a, '15 c, Chap. 111, '16 a, '16 c; Hyman, '16), including the developmental stages of the sea urchin. This demonstration has been made possible by the differences in susceptibility to various agents which are correlated with the differences in metabolic rate and protoplasmic condition at different levels of the gradient.

So far as this relation between susceptibility to inhibiting agents aiid metabolic rate has been investigated, it is as follows. In coiiccntrations of cyanides, narcotics so far as tested, acids, and, under certain conditions, alkalis, which kill, not immediately but. rapidly enough so that the organism does not, become acclimated or acquire a tolerance to them, the susceptibility varies directly with the general metabolic rate or with the rate of certain fundamental metabolic reactions. To low concentrations, tro which more or less acclimation is possible, the degree and rapidity of acclimation vary in general directly with the metabolic rate- with certain exceptions (Child, '14 b)-and within certain limits of low concentration the parts least acclimated die, while those more fully acclimated remain alive, so that in the long run the susceptibility varies inversely as the metabolic rate (Child, '13 a, '15 b, Chap. 111).

In Planaria, for example, it has been shown (Child, '13 b) that t o the higher concentrations of cyanides and narcotics the susceptibility decreases from the anterior end posteriorly, while in the very low concentrations the degree of acclimation is greatest at the anterior end (Child, '12), and in the long run susceptibility is greatest at the posterior end and decreases anteriorly.

LARVAL DEVELOPMENT IN T H E SEA URCHIX 6 7

From the metabolic point of view, acclimation consists in the gradual increase in rate of reaction in the presence of the inhibit'ing agent, and the inverse relation between capacity for acclimation and metabolic rate means that, the higher the metabolic rate, the greater the capacity for gradual increase of rate after the first inhibiting action.

Recovery after the temporary action of an inhibiting agent consists, like acclimation, in the attainment of a higher metabolic rate, but the rate attained is much higher than in acclimation because the inhibiting agent is completely removed. Where the action of the inhibiting agent has not gone so far that recovery is impossible, the rapidity and degree of recovery like acclimation vary directly with metabolic rate; the higher the metabolic rate the more rapid and complete the recovery.

If these conclusions are correct and if axial gradients a;.e present in organisms and are actual effecbive factors in development, these relations between susceptibility and metabolic rate afford n basis for the control and modification of development in two opposite directions. First, by the use of concentrations of an inhibiting agent sufficiently high to prevent acclimation to the reagent, or concentrations and periods of action sufficient to prevent recovery after temporary subjection, it should be possible to inhibit the regions of higher rate of reaction in an axial gradient to a greater extent than those of lower rate. If the rate of reaction in the apico-basal axial gradient decreases from the apical region basally, the apical region should be most inhibited, the basal least under such conditions. Second, by the use of low concentrations which permit some degree of acclimation, or by temporary action of concentrations whose effect is readily reversible, it should be possible to obtain a greater degree of acclimahion or more rapid and complete recovery in the region of high rate, i.e., the apical region, and so to inhibit its development to a lesser extent than that of the regions of lower rate. In short, it should be possible to produce, in the one case, a gradient or gradients in inhibition of development, and, in the other, gradients in acclimation or recovery. In fact, by determining proper Concentrations and times of exposure, it

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should be possible, not only to produce differential or graded inhibition or acceleration of development along an axis, but to determine the position of the greatest degree of inhibition or acceleration, either at the high or low end, the region of highest or that of lowest rate in the axis. To obtain a particular degree of differential inhibition, the procedure must of course be vaned somewhat according to the toxicity of the reagent.

The experiments on the developmental stages of the sea urchin were undertaken with these ideas in mind and with the purpose of demonstrating that the axial gradients are determining and effective factors in embryonic development. The results of the experiments have completely realized expectation and leave no doubt as to the fundamental character of the r61e played by the axial gradients in the development of the sea urchin. Some similar data on the starfish, which I hope to supplement by further experiments before publication, indicate that the relations are essentially the same there as in the sea urchin.

THE AXIAL RELATIONS I N NORMAL DEVELOPMENT

Throughout the paper the terms ‘apical’ and ‘basal: are used to designate the two ends of the apico-basal, polar, or major axis, the apical end representing the apical or ‘animal,’ the basal end the ‘vegetative’ pole of the egg. In speaking of metabolic gradients, the ends or levels of different metabolic rate in a gradient are distinguished as high and low, or higher and lower ends or levels.

According to Boveri (’01 a ,’01 b) the micromeres arise at the basal pole, and the micromere region gives rise to themesenchyme, but Garbowski (’05) has shown that polarity of cleavage and of the later stages does not always correspond to the axis indicated by the pigment ring jn Strongylocentrotus lividus. The elongation of the blastula occurs in the direction of the apico- basal axis, and the apical pole of the blastula represents the apical pole of the egg. Before gastrulation, the basal region of the blastula wall becomes thicker than the apical (fig. 1). In the gastrula (fig. 2) the apex of the conical body represents the apical end of the major axis, and the blastopore region the basal end, while the invaginated entoderm represents a still more

LARVAL DEVELOPMENT IN THE SEA URCHIN 69

basal region of the egg and blastula, and the mesenchyme the most- basal region.

The transformation of the gastrula into the pluteus begins with change from the radially symmetrical form of the early gastrula to a bilaterally symmetrical form. The basal outline of the gastrula becomes somewhat triangular instead of circular (fig. 3 A , basal aspect) and the apical region appears to shift toward one end of the longitudinal axis and becomes the oral lobe (fig. 3 B, lateral aspect). Meanwhile the apical end of the enteron unites with the body-wall at the point near the apical pole where the mouth develops, and the enteron is marked off into three regions, the oesophagus, the stomach-intestine and the rectum or anal region. Also during this period the skeletal rods appear, the body elongates and the anal arms begin to develop (fig 4, lateral aspect). Further development consists in the continued elongation of oral lobe, the appearance of the short oral arms and the elongation of the anal arms and of the body. The fully developed pluteus (fig. 5 A basal and fig. 5 B, lateral aspect) possesses a very definite antero-posterior axis, the anterior end being the broad end bearing oral lobe and arms, with the ciliated band running over the arms and oral lobe and the body margins between them, the posterior end, the opposite tapering end. Since the oral lobe represents the apical, and the anal surface the basal end of the original apico-basal axis, it is evident that this axis is no longer straight but curved, in consequence of the shifting of the apical region toward the anterior end. The course of the alimentary tract is in some degree a record of this change. In the analysis of the differential inhibitions it is essential to keep in mind this modification of the original apico-basal axial relations, as well as the antero-posterior relations of later stages.

METHODS OF EXPERIMENT

The reagents used in this series of experiments were potassium cyanide, ethyl alcohol, hydrochloric and acetic acids, sodium hydrate and ammonium hydrate in various concentrations. The eggs or embryos were placed in the solutions in

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500 cc. or 1 1. corked Erlenmeyer flasks filled almost to the cork, and solutions were renewed every twelve hours where the period of action was longer than that. It has been previously determined that development would proceed normally in such corked flasks in well aerated water, renewed every twelve hours, provided the number of eggs or embryos was not too great, and particular care was taken in the experiments that the number should not be excessive. In the experiments involving recovery after temporary action of the reagent, lots of eggs or embryos were returned to sea water a t certain intervals, with four or five changes to remove as far as possible all traces of the reagent.

THE EXPERIMENTAL MODIFICATIONS OF DEVELOPMENT

If the above statements concerning the relations between direct susceptibility, acclimation and recovery and metabolic rate, are correct, and if metabolic gradients are actual and effective factors in embryonic development, it is evident that three possibilities exist for the modification of development by the action of inhibiting agents. These possibilities are : first, direct differential inhibition of development by relatively high concentrations or highly toxic agents, in which case the degree of inhibition should vary directly with metabolic rate ; second, indirect differential inhibition through differential acclimation to lower concentrations or less toxic agents, in which case the degree of inhibition should vary inversely as the metabolic rate; third, indirect differential inhibition through differential recovery after t,emporary action of the agent.

In the forms produced by direct differential inhibition, the development of apical, anterior and median regions is more inhibited than that of basal, posterior and lateral regions, while, in the forms resulting from differential acclimation, the development of basal, posterior and lateral regions is more inhibited than that of apical, anterior and median. The forms resulting from differential recovery are, in general, similar to those resulting from differential acclimation.

The experimental results realize expectation.

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In short, these experimental methods produce two opposed types of teratological forms with various gradations between them, and the production of the two types can be controlled to a very considerable degree, the differences in susceptibility in different individual eggs and different lots being the chief limiting factors in control. For convenience the forms produced are described under the four heads : direct differential inhibition, differential acclimation, differential recovery; differential inhibition with general recovery; and consideration of the question of control follows the description. The figures of the t.era- tological forms are semidiagrammatic and are intended to show the form, axial relations, and chief structural features, details of mesenchymal distribution and slight skeletal variations being usually omitted. Wherever the distinction has seemed necessary or desirable, the middle region of the enteron has been drawn with a double contour, as its walls are thicker than those of the oesophageal and rectal regions which are indicated by single contours. All figures were drawn directly from living material.

THE FOltMS RESULTING FROM DIRECT DIFFERENTIAL IXHIBITION

The results of direct differential inhibition are most clearly seen where the development takes place in KCN, for little or no acclimation t o KCN occurs within the short period of development from egg to larva. In NH40H, development also occurs without appreciable acclimation. Some degree of acclimation to NaOH occurs during development, but in alcohol and acids acclimation occurs so much more rapidly and completely than in the other agents used, that concentrations high enough to produce a large percentage of partial or total death must be employed to produce the forms characteristic of differential inhibition.

Considering in order the various degrees of departure from the normal form, the first appreciable differential inhibition appears as a slight change in the proportion of parts in the pluteus. A comparison of figures 6 A and 623, drawn from a very slightly inhibited pluteus, with figures 5 A and 5B, (a

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normal form) shows that in the former the oral lobe is shorter and less developed, the anal arms slightly shorter and the angles of divergence between the lateral skeletal rods and between oral lobe and arms are slightly smaller than in the latter, while the length of the body from the base of the anal arm is essentially the same. In these plutei the development of the oral lobe, which represents the apical region, is most inhibited and the anterior end is smaller in both dimensions than in the normal animal, while the posterior region is fully developed. Forms such as this, in which the angles of divergence of theskeletal rods are less than the normal, are called, for convenience, narrow-angled forms. These differences may appear in different degree in different individuals of the same lot, in some the only difference from the normal being a slightly shorter oral lobe, while in others, where the oral lobe is inhibited to a greater degree, the angles of divergence, which are indices of the development of the anterior end in the direction of the minor axes, are also smaller than in the norm. Where the degree of differential inhibition is so slight, the earlier development is merely somewhat retarded without appreciable departure in its course from the norm, and the differential inhibition begins to appear only in the later stages.

A somewhat greater degree of inhibition produces forms which attain the condition of figure 7 A and B, but develop no further. Here the larva remains small, the development of the oral lobe is completely inhibited, the anal arms remain short, and the dimensions of the anterior end, as compared with more posterior regions, are much reduced, so that the angle of divergence between the skeletal rods is also reduced as compared with the normal form. The entoderm, however, still undergoes differentiation into three parts and the mouth forms. It is evident that in such cases the development of the apical region is most inhibited and the development of the anterior end more than that of the posterior end.

Where the degree of inhibition is still greater, the differential inhibiiton may appear even as early as the elongated blastula stage, as a relative decrease in apical and increase in basal di-


mensions (fig. 8). The gastrula formed from a blastula like figure 8 is flatter, i.e., apical inhibition is greater than in the normal form (fig. 9). Occasionally, in the more extreme cases, the entoderm evaginates instead of invaginating, and an exogastrula results, but in such cases, so far as my observations go, at least some of the mesenchyme cells pass into the blastocoel. Figure 10 shows the beginning of exogastrulation, and figure 11, an exogastrula. Under no conditions, however, has exogastrulation been observed in more than one or two per cent of the inhibited forms.

In the more extreme degrees of inhibition, the form of the body becomes more nearly spherical, the entodermal diff eren- tiation more completely inhibited and the skeleton more rudimentary or completely absent, although, even when they are entirely unable to form a skeleton, the mesenchyme cells are not dead, but persist in the blastocoel. In most cases where a skeleton forms, the angle of divergence between the skeletal rods is narrow. Figure 12 A and B, shows a form with rudimentary narrow-angled skeleton and figure 13, a form in which the skeletal rods are almost parallel. Figure 14 is an anenteric larva in side view with rudimentary skeleton, a form resulting from an exogastrula; figure 15, an askeletal form with differentiated entoderm, and figure 16, a still more inhibited askeletal form, in which the entoderm never develops beyond the stage of the spherical vesicle attached to the body wall. In forms like figure 16, apico-basal and other axial differences in the ectoderm never appear, and the blastopore usually closes completely, so that it is impossible to determine whether the position of the entodermal vesicle represents the basal region or not, and certain cases of differential recovery to be described below will show that the entoderm may entirely lose connection with the blastopore region. In the case of exogastrulae where the entoderm is entirely lost, the askeletal forms develop into thin walled ectodermal spheres containing scattered mesenchyme cells, and without any visible axial differentiation in any direction.

In these spherical forms all axial differentiation beyond that of the gastrula is inhibited, and it is of interest to note that in

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these cases the mesenchyme cells are scattered irregularly and do not aggregate in the basal region, as they do to a greater or less extent in less inhibited or normal forms. Evidently the localized differences in the ectoderm which supposedly determine the distribution of the mesenchyme are not present to a sufficient degree to be effective.

The most extreme degrees of differential inhibition are partial forms in which a portion of the body hzs been killed. In these cases death proceeds from the apical region, and the part which remains alive therefore consists of more or less of the basal region (Child '16 c). When such partial apical death occurs before the gastrula stage, the gastrulae are small, with a dispropor- tionately large entoderm, though not always as large as the normal (figs. 17 and IS), gastrulation itself apparently being inhibited to some degree. When partial apical death occurs after gastrulation, the result is not essentially different. These partial basal forms show no ectodermal differentiations, remain spherical, the blastopore closes, and the entoderm remains as a spherical vesicle (figs. 19,20, 21). As the figures indicate, they vary in size according as they represent a larger or smaller fraction of the apico-basal axis, and in the smaller forms, such as figures 20 and 21, the entodermal vesicle completely fills the blastocoel, because the whole of the entoderm, and only the basal portion of the ectoderm, remain alive.

In all these spherical forms, whether whole (fig. 15) or partial (figs. 19 to 21), the entoderm often loses its coherent epithelial character after several days of life without further development. In figure 22 this process is indicated. The cells become scattered about the blastocoel, and such forms are distinguishable from the anenteric forms resulting from exogastrulation, only by the larger amount of cellular material in the blastocoel. I am inclined to regard this disintegration of the entoderm as connected with its lack of functional activity, and it may be that obliteration of the metabolic gradient in the entoderm is also concerned. At any rate, this entodermal disintegration has been observed only in the inhibited forms, where the entoderm is cut off from the bpsal region and does not differentiate.


The forms shown in figures 6, 7, 12 to 16, and 19 to 21, are the final stages of development attained in the different degrees of inhibition. This does not mean that the animals die at these stages, but simply that no further development occurs, although life and more or less movement may continue for days. It is evident that the various degrees of inhibition are likewise progressive steps in the elimination of the axial relations as effective factors in development and differentiation. If the axes are fundamentally metabolic gradients this is easy to understand, for, since susceptibility varies directly with metabolic rate, the decrease in rate is greater in regions of higher than in regions of lower rate, and the metabolic gradient is therefore more or less completely obliterated by leveling down according t o the degree of inhibition. When this obliteration by leveling down proceeds to a certain point, the metabolic gradient ceases to be an effective factor in development and differentiation, and the animal becomes physiologically anaxiate, and, therefore, development and localized differentiation cease, although life may continue for a long time.

THE FORMS RESULTING FROM DIFFERENTIAL ACCLIMATION

Differential acclimation to an inhibiting agent is of course pre- ceded by a greater or less degree of differential inhibition which, however, is always less than where the action of the agent is sufficient to prevent acclimation. The effect of differential acclimation on the course of development and on the form of the body, differs in degree, according to the degree of inhibiting action and the rapidity and degree of acclimation, but the characteristic feature of the morphological modifications resulting from differential acclimation is that they are opposite in direction to those resulting from differential inhibition. The regions most inhibited in the one case show the greatest relative acceleration and over-development in the other.

Of course where acclimation is slight in degree or occurs slowly, differential acclimation may only partially obliterate the differential effects of inhibition, so that the resulting form merely shows a less extreme degree of differential inhibition than it

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would if no differential acclimation had occurred. In such cases the form is not an adequate criterion, though increase in motor activity and rate of development may indicate that some degree of acclimation is taking place. But when the axial relations of differential development are the opposite of those resulting from differential inhibition, the form is sharply distinguishable from the form produced by differential inhibition, and it is with such forms that we are at present concerned.

Where the direct inhibiting action of the agent is not very great, differential acclimation does not produce any extreme modifications of form, but appears merely as a change in the proportions and angles of divergence of parts of the plutues. Figure 23 A and B, shows a differentially acclimated pluteus in basal and lateral aspects. A comparison of this figure with figures 5 A , B, which represent the usual form of plutei raised in pure sea water shows that, in the acclimated form, the oral lobe, i.e., the apical region, is longer and broader, particularly at its apical end, the body is shorter and less slender, and the angles of divergence between the two anal arms, between anal arms and oral lobe, and between the short arms on the oral lobe is greater than in the sea water forms. Moreover, a comparison of figure 23 with figures 6 A , B, which show results of slight direct inhibition, makes the opposite character of differential inhibitory and differential acclimatory changes strikingly evident.

Not infrequently in these wide angled forms, the angle between the anal arms is somewhat wider than between the posterior skeletal rods, as in figure 24. This indicates a greater degree of disproportion in development between the anterior region and the more posterior levels than in figure 23, i.e., breadth of the anterior region is increased out of proportion to that of other levels of the antero-posterior axis, and the angle between the arms is therefore wider than that between the more posterior portions of the skeleton.

In KCN and NH,OH, where acclimation is relatively slight and occurs so slowly that development is usually completed before the differential effects of acclimation become marked, even the slight changes of form shown in figure 23 do not usually


occur, but in alcohol and hydrochloric and acetic acids acclimation occurs much more rapidly and to a much greater degree, so that differential acclimation is often evident in the early gastrula stage. Consequently, with these agents much more extreme modifications of form can be produced through acclimation. NaOH is intermediate between these two groups as regards the forms resulting from differential acclimation.

The earliest marked indications of differential acclimation which have been observed occur in alcohol and acids, and consist in increase in the relative size and growth of the apical. region of the gastrula. In acids this is very commonly a tapering pro- longation of this region (fig. 25), while in alcohol it is more often rounded (fig. 26 B) , but both forms may appear in both agents. Irregularity of outline and roughness of the external surface of the basal region of the gastrula (figs. 26 A , B) , is particularly characteristic of the action of alcohol, though often produced to a lesser degree by the acids. It is evidently due to a decrease in the normal epithelial coherence of the cells in this region, but as development and acclimation proceed, i t almost or quite dis- appears.

Where the apical region undergoes a relative acceleration in stages as early as the gastrula, disproportion between it and other part.s increases as development proceeds. Figures 27 and 28, are side views of prepluteus stages showing this disproportion. As compared with the normal form at this stage (fig. 3 B) the apical and anterior regions are relatively greatly over-developed, and basal and posterior regions, including skeleton and arms are greatly under-developed. These alterations are exactly the opposite of those resulting from differential inhibition (fig. 7 B). In the acids, where acclimation occurs more rapidly and the differential features are more marked than in any other agents used, the most extreme modifications of t8his sort occur. Figures 29 and 30, A basal, B lateral aspect, show cases from acid in which there is enormous over-development of apical, as compared with basal regions, and in which the anterior region is so much over-developed that the arms approach or attain an angle of 180°, while in more posterior regions the angle of divergence,

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although wider than normal, is not as great as that between the arms. In these cases the effect of differential acclimation has been extreme in the apical and anterior regions, but relatively less in more basal and posterior parts. Figures 31 to 36 show the final stages of development attained in the more extreme degrees of differential acclimation to acids. Figure 31 A is the basal and figure 31 B the lateral aspect of a larva with large oral lobe, small body and no arms. The body is short and broad and very wide angled. Evidently apical and anterior regions are relatively over-developed, basal and posterior relatively under- developed. In figure 32 A (basal) and B (lateral), these modifications are more extreme. The oral lobe is relatively longer, the body shorter and broader, and the angle of divergence wider. Figure 33 shows a still more extreme degree of modification, A being the basal, B the lateral and C the anterior aspect, and figure 34 shows the most extreme modification possible in this direction, A , B, and C being, as before. basal, lateral and anterior aspects. Here the oral lobe is larger than the body, and the skeletal rods are united to form a single straight rod lying transversely, and passing anterior, instead of posterior, to the anus. The angle of divergence between the rods is here 180". These forms in figures 31 to 34 differ from those in figures 29 and 30 in that the angles of divergence of the skeletal rods are uniform throughout, instead of being wider anteriorly than posteriorly. This means that in figures 29 and 30 the differential effect of acclimation is more extreme anteriorly than posteriorly, while in figures 31 to 34 it has extended far enough posteriorly to determine a uniform angle of divergence of the whole skeleton, instead of a wider angle anteriorly than posteriorly. In figures 35 and 36, A , B, C, forms similar in outline and axial relations, but without skeleton, are shown.

It is evident that all these forms are the result of relative differential accelerations along the axes. Acclimation has made the slope of the axial gradients steeper; i.e., the regions of highest metabolic rate in each gradient have now a relatively much higher rate, and the regions of lowest rate a relatively much lower rate then under normal conditions. Consequently the greatest de-

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gree of over-development, as compared with the norm, is in the regions of highest rate, i.e., apical and anterior, and apparently also median; and the greatest degree of under-development is basal, posterior and apparently lateral. In consequence of these changes in relative metabolic rate along the axes, the larva is transformed from the antero-posteriorly elongated normal form (figure 3 A ) into a broad, transversely flattened, form with short antero-posterior axis ; and all stages of this transformation appear.

In cases where the degree of inhibition is somewhat greater, differential acclimation in acid gives rise to forms like figure 37, which shows basal ( A ) , lateral (B) , and anterior (C), aspects of a characteristic acid form. In this larva the ciliated band, which, in the normal larva and the less modified forms, extends as a continuous band around the margins of the anteriw end and over the oral lobe and basal arms, is differentiated only in the apical and basal regions, as indicated in figure 37 by the shaded bands. The apical portion of the band extends around t,he rudimentary oral lobe and a short distance basally, while the basal portion extends from the medial anterior region posteriorly more than half way around the body, and the lateral portions, which normally connect these apical and basal portions, are not present. In fact, the basal portion of the ciliated band in such cases forms a more or less complete ring around the basal region. This condition recalls the condition in the more primitive type of echinoderm larvae where several ciliated bands surround the body. Apparently in such cases as this, where the antero- posterior a.xis and bilaterality are practically obliterated, the local metabolic conditions determine that the ciliated band shall develop around the basal region, instead of over the arms and around the lateral margins of the anterior region. This condition also appears frequently in recovery forms (pp. 85, 87, 88).

When the direct inhibiting action of the agent is somewhat, more lasting, and acclimation occurs somewhat more slowly, as in alcohol, the changes resulting from differential acclimation are, so to speak, superimposed on the changes resulting from differential inhibition. In such cases, therefore, various combina-

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tions of differential inhibition and differential acclimation appear. The form shown in figure 38, for example, is, in its basal aspect ( A ) ; a characteristic case of differential inhibition, resembling figure 12 A , with the inhibition decreasing from the anterior to the posterior end, so that the body is relatively nar- rower and the angle of divergence of the skeletal rods less than in the normal (fig. 3 A ) .

The side view of this larva, fig. 38 B, is very different from that of the differential inhibition, figure 12 B. The oral lobe is large, with a rounded knob on its apical end, and the ciliated band develops only in this apical region. Great elongation in the apico-basal axis has occurred, and the enteron is attached a t the mouth region, but not at the anal, and the rectal portion is absent.

Figure 39 is a side view of a form in whieh differential acclimation follows a somewhat greater degree of inhibition and consists chiefly in the apical outgrowth and the broadening of the anterior end.

The observation of many similar cases has shown that the course of development in cases of this sort is as follows: during the earlier stages of development the action of the agent is directly inhibitory, with the usual differential effect, but, at about the gastrula stage, differential acclimation begins to be apparent as a relative acceleration of development in the apical region, as in figures 25 and 26 R. From this apical region the rela- t,ive acceleration progresses basally, but is more marked in anterior than in posterior, and apparently also more in median than in lateral regions. The basal region undergoes acclimation so much less rapidly and less completely than the apical that it shows no secondary changes before development comes to a standstill in the solution.

When the apical elongation begins the entoderm remains attached to the region where the mouth is to form, and it may break its connection with the blastopore region, either through closure of the blastopore (which often occurs in inhibition) or by actual rupture and so is carried apically and remains closed posteriorly, arid the rectal region is absent. Tn short these larval


forms show differential acclimation in the more apical regions and differential inhibition in the more basal.

Figures 40 t o 46 show the final stages of differential acclimation in alcohol after still greater degrees of inhibition. In all figures the apical end is uppermost, but the antero-posterior axis has been obliterated to such an extent that in most cases i t is impossible to distinguish anterior and posterior regions. The most apical portion of the ciliated band is differentiated in figures 40 to 43, but in figures 44 to 46 differentiation ceases a t an earlier stage. The different positions, degrees of differentiation, and relations of the entoderm depend upon the relations between the time when apical acclimation began, upon the degree of differentiation of the entoderm and upon the degree of direct inhibition. Where the direct inhibition does not bring about closure of the blastopore (fig. 15), the entoderm may separate between stomach-intestine and rectal regions, the latter re- maining a t the basal end and undergoing degeneration (fig. 40), or elongating and maintaining its normal basal connection (fig. 41). In other cases the blastopore closes completely, and the entoderni loses connection with the blastopore region, but remains in contact with the apical region of the body wall, and is carried with it when elongation or enlargement occur. Under these conditions it may differentiate completely, or the rectal region (fig. 40), or mouth and oesophagus (figs. 41, 42) may be absent, or lastly, it may remain a rounded vesicle (figs. 43 to 46).

There the entoderm remains a closed vesicle it often undergoes degeneration after a few days (figs. 47, 48) as in differential inhibition (p. 74), leaving anenteric forms which may remain alive for several days longer.

Even though no skeleton develops in these forms, more or less aggregation of mesenchyme in the basal region occurs as in normal development, while, in the more extreme degrees of in7 hibition without acclimation, the mesenchyme remains irregularly scattered in the blastocoel. Evidently in acclimation the apico-basal metabolic differences are sufficient to constitute effective factors in determining the localization of the mesenchyme, while a sufficient degree of inhibition makes them ineffective.

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If this localization is chemotactically determined, as is commonly believed, then we may say that a certain difference in metabolic rate along the apico-basal axis is necessary as a basis for the differential chemotactic action.

Attention must be called to the possibility that, in some of the forms shown in figures 40 to 48, the original apical region has been killed before acclimation, and that the apical outgrowth is a reconstitution from more basal regions. It is perfectly certain from the observations on earlier stages that forms like figures 40 t o 43 may develop without any apical losses, but it is probable that at least in some cases forms like figures 44, 46 have lost some apical cells. In forms such as figures 38, 39, 40 to 48, which are very characteristic of acclimation in alcohol, the acclimation involves chiefly the more apical regions of the body, while in the basal regions the effect of inhibition persists. The difference between these forms and the acid forms (fig . 41 to 37) is due to the fact that in acids differential acclimation begins earlier in the regions of highest metabolic rate of the various axes and is more complete than in alcohol, therefore the extremes of the resulting form changes are greater than in alcohol. In alcohol differential acclimation overcompensates the effects of differential inhibition only in the more apical regions of the apico-basal axis, and differential acclimation along the antero-posterior axis plays little or no part in determining the form, while in acids the differential effect is merely greater in the apico-basal axis and is also marked in the antero-posterior and medio-lateral directions.

The lower limits of differential acclimation are reached in the partial basal forms where a considerable portion of the apicad region of the ectoderm has been killed by the direct inhibiting action of the agent. Without acclimation, such forms remain spherical, the blastopore usually closes and the entoderm becomes a closed vesicle in the blastocoel (figs. 19 to 21), but, under conditions which permit some degree of acclimation, they give rise to forms like figures 49 t o 53. In these a small apical outgrowth may occur with the mouth at its base, presumably on its anterior side (fig. 49); the mouth may lie at the apex of

LARVAL DEVELOPMENT IN THE SE.4 URCHIN 83

an apical elongated region (fig. 50); an apical outgrowth niay occur and mouth and oesophagus fail to differentiate (fig. 51); or mouth and oesophagus may develop in the absence of an apical outgrowth (figs. 52,53). Usually in these cases theenteron is not connected with the blastopore region, and the rectal portion does not differentiate (figs. 49 to 51, 53) , but occasionally in acids complete differentiation of the entoderm has been observed (fig. 52). Except for the case of figure 52, which has been seen only in acclimation to acids, these forms occur in both alcohol and acids.

In these cases of limited acclimation following a rather high degree of differential inhibition (figs. 40 to 53), the antero-posterior axis and bilaterality have been almost or quite obliterated as effective factors in development and, except for the position of the mouth in some cases (figs. 40 and 49), do not appear in differential acclimation. The larvae remain almost complete1:r radially symmetrical and move with the apical end in advance as long as they live. Only the apico-basal axis remains effective, and the metabolic gradient, which constitutes this axis, has been levelled down to such an extent that it is much less effective in determining and localizing differentiation than under the usual conditions. These partial acclimations, as well as the more extreme differential inhibitions, show very clearly that the minor axial gradients can be leveled down and obliterated t o such an extent that they do not reappear, while the apico-basal gradient may still remain effective to some extent. This can only mean that these minor metabolic gradients are less permanently recorded in the protoplasmic substratum (Child, '15 c, pp. 33-35), and this in turn must mean that the high ends of these minor gradients represent lower metabolic levels than the high end of the apico-basal gradient, i.e., the apical region. This conclusion is in agreement with all the facts. If the apical region is the region of highest metabolic rate in the individual, it follows of course that the high ends of gradients in other directions must have a lower rate than the high end of the apico- basal gradient; moreover, the death gradient is much more distinct in the apico-basal axis than in other directions (Child,

84 C. M. CHILD

’16 c), and the minor axes are less effective as factors in localization and differentiation, or become effective later than the major axis. This differential obliteration of axial gradients is one o f the most significant results of these experiments, and the forms in which only the apico-basal gradient remains as an effective factor in development approach in certain respects the more primitive types of echinoderm larvae.

THE FORMS RESULTING FROM DIFFERENTIAL RECOYERI’

In recovery after temporary action of an inhibiting agent, a, distinction must be made between the general and the differential effect. Where the effect of the inhibiting agent has not been so great that recovery is impossible, all parts of the body undergo an increase in metabolic rate after return to water. This is the general effect. The differential effect appears in the differences in rate and degree of recovery along the axes and is like the differential effect in acclimation; i.e., the higher the metabolic rate, the more rapid and complete the recovery, but since in the developmental stages of the sea urchin recovery, like acclimation, follows and is superimposed on the differential effects of inhibition, the forms resulting from differential recovery, like those resulting from differential acclimation, may show various com- binations of differential inhibition and differential recovery. Since recovery involves not merely the differential changes in metabolic rate, but a great increase in rate in all parts nft,er removal of the inhibiting agent, development may proceed farther, if the temporary inhibiting action be not too great, than where the agent is present throughout. This difference is responsible for certain interesting features of recovery which will be considered below.

Unfortunately the data concerning recovery are not complete. The close of the breeding season of Arbacia put an end to the experiments when only KCN and alcohol had been used in this way, but, since the results with these agents are similar to the results of differential acclimation, there is every reason to believe that the same would be true for alkalies and acids.


Where inhibition is slight and limited to the earlier stages of development, differential recovery may appear as elongation of the apical region in the gastrula (figs. 54, 55) or in prepluteus sta.ges (fig. 56). Such forms give rise to plutei like the acclimation plutei of figures 23 and 24 with very large and long oral lobes, wide angles of divergence, and short body.

From forms of this sort, the gradation, through various com- binations of differential inhibition and differential recovery, to forms which show no differential recovery is complete. Cer- t8ain features of these forms require some consideration.

Where the inhibition is so great that minor gradients are almost or quite obliterated, while the apico-basal gradient still remains an effective factor in development, recovery produces a series of forms resembling the differential acclimation forms of figures 37 and 40 to 53. Figures 57 to 73 show examples of diff erent,ial recovery after a considerable degree of inhibition by cyanide. In all cases the minor axes are almost or quite oblitcrated, but there is usually elongation in the apico-basal axis, and in many cases (figs. 57 to 64) an apical outgrowth, a rudimentary oral lobe, on which the apical portion of the ciliated band differentiates. Frequently also the basal portion of the ciliated band differentiates as a more or less complete basal ring (figs. 57 to 62, 65 to 72) as in figure 37. Here, as in differential acclimation (p. 79), the differentiation of the basal portion of the ciliated band as a partial or complete basal ring is associated with the almost complete obliteration of the antero-posterior axis and of bilaterality by the differential inhibition. This basal ring appears much more frequently in recovery than in acclimation, because, after a given degree of inhibition, differentiation proceeds somewhat further when the animals are returned to water than in the continued presence of the inhibiting agent. The development of the apical outgrowth and the apico- basal elongation are features of differential recovery, but the basal ciliated ring is a consequence of general recovery (or acclimation, fig. 37) following a degree of differential inhibition which almost or quite obliterates the minor axes. Both apical outgrowth and basal ciliated ring may be present (figs. 57 to 62)

86 C. M. CHILD

or the basal ring (figs. 63 to 64), the apical outgrowth (figs. 65 to 72), or both (fig. 73) may be absent. It is of interest to note that those forms with well developed basal ciliated ring move with basal end in advance almost as frequently as in the opposite direction.

In some of these forms the entoderm shows normal relations and undergoes normal differentiation (figs. 57, 65, 66), but more usually the blastopore closes completely, and the entoderm loses connection with the basal region, but remains attached to the apical region. Under these conditions a mouth may develop and differentiation of the entoderm into the three parts may occur (figs. 63, 67 to 69); and in such cases the rectal region either ends blindly (figs. 63, 68, 69), or occasionally a new anal opening arises, apparently wherever the rectal region is in contact with the body wall (fig. 67). I n other cases mouth, oesophagus, and stomach-intestine are present, but the rectal region fails to develop or, perhaps, becomes separated from other parts and degenerates (fig. S l ) , as in some cases of acclimation (see p. 81). In still other cases mouth and oesophagus may be absent; but stomach-intestine and rectal region may differentiate (figs. 58, 59, 73), or the entoderm may show little or no trace of regional differentiation (figs. GO, 64, 70, 72). Occasionally the entoderm remains in contact with the blastopore region, though completely closed and without regional differentiation (fig. 64), and a few individuals with evaginated entoderm (figs. 62, 71) resulting from exogastrulation (p. 73) occur.

These various degrees of development and relations of the entoderm depend on the dcgree of inhibition, the time when apico-basal elongation begins and perhaps on other factors as well. In the more extreme degrees of inhibition the blastopore usually closes completely and the entoderm becomes a closed vesicle. If acclimation or recovery occurs in such cases the entoderm usually becomes attached to the apical region of the body wall, and its further development depends on conditions in this region of.the body wall and on the degree of entodermal inhibition. I n some cases, where the blastopore does not completely close, the entoderm may reach the apical region and retain


its normal relations, or rupture may occur between stomach- intestine and rectal region, the latter being left in connection with the blastopore and usually degenerating. The exact his- tory of each particular case can be determined only by isolation and continuous observation of individuals, and this I have not attempted.

Although no skeleton develops in forms of the character of figures 57 to 73, there is in nearly every case, as in similar acclimation forms, a distinct aggregation of mesenchyme cells in the basal region where the skeleton normally arises, and the conditions which determine it are evidently the same as in acclimation (p. 81).

It is probable that at least many of the forms in figures 57 to 73 have lost some part of the apical ectoderm by apical partial death, resulting from the direct differential action of the agent, and that the apical outgrowth is therefore, in the strict sense, a reconstitution of the apical region. It is certain that such forms may develop, both where a part of the apical region has died and where there have been no apical losses by death, so that the question, whether the apical outgrowth is a reconstitution of the apical end from cells which were originally not apical, has little significance. Physiologically, it is a reconstitution, whether apical losses have occurred or not, for with a sufficient degree of inhibition the apical cells themselves are incapable of developing an oral lobe, but when their metabolic rate rises to a certain level, in consequence of differential acclimation or recovery, they become capable of such development, and, since they acclimate or recover more rapidly than cells below them, the development may take the form of a local reconstitutional outgrowth.

These cases of recovery are of the same general type as the cases of differential acclimation in figures 37, and 40 to 46. In both groups the antero-posterior axes and bilaterality are almost or quite obliterated, and the apico-basal axis remains as the chief or only effective factor in orderly development.

9 s noted above (p. 79) forms of this kind recall, in various ways, the more primit8ive types of echinoderm larvae. In the

58 C. M. CHILD

crinoid larva, for example, the blastopore closes completely, the entoderm forms a closed vesicle in the blastocoel and the anus breaks through at another point. All of these conditions, even the new anus (fig. 67), appear in these recovery forms of Arbacia. Likewise the basal ciliated ring recalls the ciliated rings of the crinoid larva. The facts suggest that in the primitive echinoderm larva, the apico-basal axial gradient is not as well developed as in Arbacia, and the antero-posterior and bilateral gradients are very slight, or become effective only in later stages. By means of differential inhibition, which decreases the slope of the apico-basal gradient and almost or quite obliterates the minor gradients, we produce axial relations which resemble, in certain respects, those of the primitive forms in which the axial gradients are less developed; i.e., less permanently recorded in the protoplasm and therefore less effective as factors in orderly development.

Figures 74 to 81 show the lower limits of differential recovery. In all, except perhaps figures 74 and 75, there has been some apical loss. In figures 74 ( A , anterior, B, lateral aspect) and in figures 75 to 77, all recoveries after alcohol, the apical outgrowth develops the ciliated band characteristic of the oral lobe. In recovery after KCN, the ciliated band usually does not differentiate where the degree of differential recovery is so slight. Figures 78 to 81 show cases in which no oral lobe is differentiated, but a mouth develops apically on an elongated apical region (figs. 78, 79), or the body remains spherical and thedevelop- ment of the mouth is the only evidence of differential recovery. The development of the entoderm shows the same variations as in figures 57 to 73. These forms are essentially similar to the cases of differential acclimation in figures 49 to 53.

The various degrees of differential recovery in figures 57 to 81 are the final stages of development attained. Such larvae may live for a week or two and may show marked motor activity, but they do not develop further. The only reason which can be assigned for this failure to resume development, is incom- plete recovery, and this means that the metabolic gradients have been obliterated or levelled down to a greater or less degree


by the differential action of the inhibiting agent, and that recov- ety does not completely restore the usual metabolic relations along the axes; consequently the metabolic differences are less effective in determining localization and differentiation than in the normal animal, or they are effective only in the apical region, where recovery is most rapid. It is evident that energy for development is available, for, in general, the greater the degree of inhibition of development, the longer the life after recovery before the animals die of starvation. It is not then that energy, but rather that the pattern or plan of energy distribution which the axial metabolic gradients afford is altered or more or less completely obliterated.

FORMS RESULTING FROM DIFFERENTIAL INHIBITION WITH GEN- ER4L RECOVERY

Where the degree of inhibition is so great, the effect so persistent, or the period of inhibition so late in development that differential recovery does not completely compensate or over- compensate the morphological effects of inhibition, the general recovery which follows return to seawater often serves to bring out the differential effect of inhibition on the form and proportions of the larva more clearly than does continued action of the inhibiting agent. This is simply because development always advances somewhat farther after the animals are returned to water, and so the differential inhibitions may be carried to later stages, than in the presence of the inhibiting agent.

High concentrations of KCN, acting for a sufficiently long period at or before the gastrula stage, give particularly interesting results in this respect, because the inhibiting effect of high concentrations of KCN is relatively persistent and is therefore less completely compensated by differential recovery, but there is no doubt that similar results can be obtained with NH40H and NaOH, and even with acids, if the degree of inhibition be sufficient and the period of inhibition late enough in development to prevent the occurrence of differential recovery before the axial relations are fixed.

90 C . M. CHILD

Narrow angled plutei like figures 6 A and B, are very characteristic results of this procedure, but more extreme alterations of the relations of parts are also of frequent occurrence. Figures 82 to 87 ( A basal, B lateral aspect), show characteristic cases. In all of these the apical region is more inhibited than the basal, but the more complete development of skeleton and arms brings out more clearly the modification of form than where the inhibiting agent is present throughout development. In figure 13, a case of differential inhibition with practically parallel skeletal rods was shown. Figure 82 A , B shows the type of pluteus produced where such a degree of inhibition is the result of temporary action of a high concentration followed by return to water. Here the longitudinal rods and arms are parallel; i.e., the metabolic differences between anterior and posterior regions have been obliterated to such an extent that both are of the same width. In figure 8 3 A , B, a somewhat different modification occurs in that the two anal arms are fused to forma flat, tapering structure and the arm rods approach each other anteriorly, while the posterior portions of the skeleton still show some divergence. Figure 84 is another case of fused arms, but with parallel posterior skeletal rods, figure 85 A , B, shows a still more complete fusion of the arms and figures 86 A , B, and 87 A , B, cases in which the skeleton is median in position. In figure 86 the rectal region and anus are absent, and in figure 87 the entoderm evidently has lost connection with the blastopore region, but is fully differentiated, and the rectal region is in contact with the posterior body wall, though an anal opening could not be found.

These forms show the changes in position and relations of parts resulting from the differential effects of inhibition on the minor axes. With decreasing metabolic difference between anterior and posterior ends, the skeletal rods become more or less nearly parallel, and, in the transverse direction, the inhibition is evidently greatest in the median region, for the width of the body decreases, and structures, normally lateral, approach the median line and finally become median as the degree of inhibition of normally median parts increases.


Such larvae as these show the extremes of modification in the direction opposite to the differential acclimations in figures 29 to 36, and the extremes in the two groups represent the limits of possibility. In the one case, figures 82 to 87, parts, normally lateral, approach the median line, while in the other, figures 29 to 36, parts normally longitudinal become transverse. And yet these extreme differences in form are brought about by altering the metabolic differentials along the axial gradients, in the one case by decreasing the differences in metabolic rate along the axes through differential inhibition, in the other by increasing these differences through differential acclimation.

One other interesting feature appears very frequently in cases of recovery, particularly after KCN, where the inhibition has not been extreme, but the differential effect is not fully compensated by differential recovery. This is an extreme development of skeletal structure as indicated in figures 88 to 90. The skeleton of the anal arms in such cases is composed of many partially fused rods, the basal part of the body-skeleton and the posterior spiny enlargements may be of enormous size (figs. 88, 89), and additional short rods may arise from the regions of junction of the different rods, and even at the median junction of the two transverse basal rods, as in figure 89. Figure 90 shows a mul- tiplication of arm rods in a more inhibited form. No attempt has been made t o figure the more extreme cases of this skeIetal over-development. In some the body seems to be largely filled with skeletal structures, and very aberrant shapes often result from the outgrowth of rods in various directions. In such cases skeleton development seems to have run wild. This skeletal over-development is undoubtedly a result of differential inhibition, but of course occurs only in cases where general recovery is brought about by return to water after temporary inhibition.

It has already been pointed out that the susceptibility of the mesenchyme cells is relatively low, as their origin from the basal region of the embryo might lead us to expect. This being the case, they are less affected by slight temporary inhibition than are the more susceptible parts, and therefore, when the metabolic rates of all parts are raised on return to water, the

92 C. M. CHILD

metabolic rate of the mesenchyme cells is relatively higher, as compared with other parts, than in uninhibited animals. Be- cause of this difference, they are able to obtain more nutrition from other parts and so to undergo more growth and division and, finally, to give rise to a larger amount of skeletal substance than normally. The normal metabolic relations between the mesenchyme and other parts have been altered t o the advantage of the mesenchyme cells by the differential inhibition. Since the mesenchyme rises from the basal region of the egg, this effect of differential inhibition upon the mesenchyme is merely a special case of differential inhibition along the apico basal gradient, the mesenchyme cells (or the region from which they arise), being less inhibited because of lower metabolic rate. Since the mesenchyme cells do not remain a part of the apico basal axis, but are distributed in certain relations to other parts, the effect of differential inhibition in this case is simply an over-development of the skeleton to which they give rise.

THE CONTROL OF THE DIFFERENTIAL MODIFICATIONS

Eliminating, as far as possible, changes of temperature, constitution of sea water and other variable conditions, the control of the results with any one of the agents used is a mztter of concentration, stage of development at which the inhibiting action begins, and, in the case of temporary action of the agent, the length of the time of action. Where the agent begins to act a t a sufficiently early stage of development, higher concentrations produce differential and general inhibition ; lower concentrations, differential acclimation, and the shorter the period of action the higher the concentration required t o produce a particular modification. In experiments on differential inhibition and differential acclimation, all six agents,-KCN, C2H50H, HC1, CH,COOH, NH,OH, and NaOH-were used, but in the study of differential recovery after temporary action, only KCN and alcohol were employed, for the close of the breeding season of Arbacia terminated the work before the data could be completed.


As regards general character and direction of modification of form, no specific differences appear in the action of the different agents used, but the limits of differential modification along any particular axis differ somewhat with different agents, because the relations between differential inhibition, acclimation and recovery are somewhat different for different agents, presumably because they act on the living protoplasmic system to different degrees or in different ways. With KCN, for example, all degrees of differential inhibition are readily produced by either continuous or temporary action, but KCN is so highly toxic to living protoplasm, and its effects are so persistent that, even in low concentrations, the degree of acclimation during the period of development is much less than with alcohol and acids, and, even in recovery after temporary action, if the concentration of KCN is high enough to produce any marked degree of differential inhibition, this is not usually entirely compensated by differential recovery. In other words, the differences in metabolic rate along the axial gradients are more completely and more permanently levelled down and obliterated by KCN than by alcohol and acids. KCN then is the most satisfactory agent among those used for producing axial differential inhibitions of development, free from, or but little complicated by differential acclimation or by differential recovery.

While it is much less powerful as a protoplasmic poison than KCN, it is much more effective in producing differential inhibition than differential acclimation, because acclimation occurs so slowly that development is either completely arrested by differential inhibition, or, in low concentrations, is completed before any great degree of differential acclimation occurs.

NaOH acts distinctly as an inhibiting agent on these marine forms, though higher concentrations than of NH40H are necessary to produce a given differential effect. Acclimation occurs more readily than to NH,OH and consequently greater differential effects of acclimation on the form and proportions are possible with this agent than with either KCN or NH40H.

Next to KCN in this respect stands NH40H.

94 C. M. CHILD

Alcohol and acids’ act as inhibiting agents in sufficiently high concentration, but are less effective in producing differential inhibitions of development than any other agents used, because in almost any concentrations which do not actually kill, acclimation begins within a very short time, and, by the time development has reached the limit determined by the conditions of experiment, the primary differential inhibition is compensated or in most cases over-compensated to a high degree by differential acclimation. Acclimation to acids occurs even more rapidly and to a greater extent than to alcohol. Consequently, with alcohol and acids, the more extreme types of differential inhibition are best obtained by the action of high concentrations, beginning at the blastula or gastrula stage instead of at the beginning of development, for this procedure leaves little time for the occurrence of acclimation. Alcohol and acids, acting from the beginning of development, are the most effective of all agents used in producing the more extreme types of differential acclimation, and the acids are somewhat more effective than alcohol. The differential effects of recovery after the temporary action of alcohol are, like those of KCN, similar in character to the differential effects of acclimation and there is every reason to believe that the same is true of acids.

It is evident from these facts that the different reagents used may be arranged in a series according to their differential effects on development and larval forms. The agents which are most effective in producing the differentially inhibited types of form are least effective in producing the types of form characteristic of differential acclimation, and vice versa, and between the extremes of KCN and acids, NH40H, NaOH and alcohol may be placed in the order given. So far as the observations go, the relations as regards differential recovery after temporary action are the same as for differential acclimation. These differences in action of different agents depend upon the rapidity and degree of reversibility of their effects on protoplasm with either continuous or temporary action. I am inclined to believe that these differences in effect upon development form and proportions may be regarded as constituting to some extent a cri-


terion of the toxicity of the different agents. The more toxic the inhibiting agent, the less rapidly and completely are its differential inhibiting effects reversed, either in its presence or after its action.

As regards the relation between the stage of development at which the inhibiting agent begins to act and the concentration or period of action required for a particular effect, it is evident that, since susceptibility increases very greatly during early development up to the gastrula stage (Child, '15 b, pp. 412418), earlier stages require higher concentrations or longer periods than later stages for the production of a given differential effect. Moreover, since the later the stage at which action begins the less time there is for differential acclimation or recovery, it is evident that the more extreme degrees of differential acclimation and recovery are most readily produced by action on the earlier stages. Where the action of the agent begins at the beginning of development and continues throughout, the result, of course, depends merely on the concentration and toxicity of the agent, and the same is true for action beginning at any particular stage, though, as already noted, the later the stage of development up to the gastrula, the lower the concentration necessary to produce a particular effect.

As regards concentration, it should be noted that in the case of KCN, when the period of action is only a few hours, the physiological effect does not increase proportionally to the concentration above a certain limit. For periods of four or five hours or less, for example, the physiological effect of KCM m/100 is not very much greater than that of m/1000, but with lower concentrations and with longer periods of action, the effect is much more nearly proportional to the concentration. No attempt has been made to determine the factors concerned in these relations between concentration and physiological effect of KCN solutions, but there are several possible factors, viz., increase in alkalinity, decrease in dissociation, with increase in concentration, the time necessary for penetration and perhaps others. Whether similar relations exist for very high concentrations of other agents used, has not been det,ermined. These are ques-

96 C. M. CHILD

tions of interest, but not directly connected with the chief purpose of the investigation.

One variable factor in the results, viz., the individual differences in susceptibility in different eggs of the same female and of different females cannot be controlled in these experiments. In consequence of these differences the differential effect of a particular concentration and period of action of a particular agent is different in degree in different individuals and in different lots of eggs. Since large numbers of individuals are used in each experiment a certain range of variation in the resulting forms appears, but this is not great enough to obscure the general effects. Some of the characteristic data for the different agents used follow.

Potassium cyanide

In the most extensive series of experiments KCN was used as the inhibiting agent, both continuously during development, and for short periods. The forms produced show differential inhibition of greater or less degree as the characteristic feature, and the degree of differential acclimation or recovery is slight. The differential effect of KCN on the axial gradients is very persistent, and, even though general recovery may occur on return to water, there is but little differential recovery during the period of larval development. The following data will serve to indicate the range of concentrations and periods of action.

Eggs placed in KCN m/50000 at first cleavage and kept in this concent,ration continuously, develop slowly to the blastula stage where in some lots of eggs 25 to 30 per cent die completely, apicd end first, while in 25 to 40 per cent death begins apically, but more or less of the basal region remains alive. In other lots practically all remain alive, at least basally, but in a larger or smaller percentage more or less apical death occurs. The final stages attained show all degrees of differential inhibition, but only rarely the slightest indications of differential acclimation.

Development from the first cleavage in KCN m/100000 gives little or no partial or total death and the forms produced show in most cases only slight differential inhibition like figure 6. In


HCN m/200000 the early stages of the plutei may show a slightly inhibited oral lobe, but later stages are usually normal in form. Development in concentrations higher than m/50000 usually ceases at the blastula stage and death occurs, the apical region dying first, and concentrations below m/200000 have no appreciable effect on development except, perhaps, a slight retardation.

In recovery after the temporary action of the higher concentrations of cyanide there are, of course, many cases in which the more apical regions are killed and only a larger or smaller part of the basal region recovers. Such cases may undergo more or less reconstitution, according to the stage of development, and may produce plutei. The following data give some idea of concentrations and periods of action.

Unfertilizedeggs, 3 hours in KCPI; wz, 1000, then washed and fertilized. Practically all develop normally and produce normal plutei. Xny differentid inhibition which may have occurred in earlier stages is comepensated by differential recovery.

Unfert,ilized eggs 64 hours in KCN m 1000, then washed and fertilized, 60 to 70 per cent develop, many with abnormal cleav- ages. In nearly all a lzrger or smaller portion of the apical region dies and is lost before the gastrula stage is reached; i.e., such portions have been so far inhibited that they :ue incapable of recovery.

The partial forms undergo more or less reconst,itution and the resulting forms show all degrees of differential inhibition with general and some degree of differential recovery superimposed (figs. 6, 57 to 73 , 82 t o 90) and, as the extreme type, spherical basal forms without rcconstitutioii (figs. 19 to 21).

1000, then washed and fertilized: 40 to 50 per cent show sonic development, but 20 t’o 30 per cent of these die before the gastrula &age, and more or less I O ~ S o f tlic apical wgions occurs in others. The forms produced show the saiw range of variety as in the preceding lot, but thc percentagc of the more extreme types of differential inhibition with less differential recovery is greater.

In these cases of inhibiting action of KCN before fertilization, the period of recovery is the whole period of development,

Unfertilized eggs 8; hours in KCN

JOURNAL OF YORPHOLOQY, VOL 28, NO. 1

9s C. M. CHILD

aid during this period the lesser degrees of differential inhibition may be more or less completely compensated by differential recovery, so that the final form may approach the norm, although, in the earlier stages, there is marked differential inhibition or even apical loss. Where the degree of inhibition and therefore the apical losses are greater, differential recovery brings about more or less apical reconstitution, but in the most extreme cases which remain alive and develop, there is no reconstitution, but only a partial basal form is produced. In consequence of these various possibilities, the range of variation in form in a single lot of eggs is greater than where the inhibiting agent acts on later stages.

Eggs placed in KCN m/20000-m/10000 at the beginning of cleavage usually continue to develop slowly, reaching the early blastula stage in 12 to 18 hours, but by the time they reach this stage their susceptibility has increased (Child, '15 b, pp. 412- 418) to such an extent that further development is inhibited. If returned to water at this time a considerable percentage of total and of partial apical death occurs, and the final stages show all degrees of differential inhibition, with differential recovery in the form of apico-basal elongatioii and apical reconstitution (figs. 57 to 81) in a varying percentage. The same forms niay be produced by a few hours in KCN m/10000 at the blastula stage. In fact, for any concentration which is low enough to permit development t o proceed to the blastula stage, the effect is essentially the same, whether the eggs are placed in KCN at early or late cleavage and left there until they reach the blastula stage, or are placed in the same concentration at the blastula stage.

Late cleavage or early blastula stages in KCN m/10000 never develop beyond the blastula stage. On return to water after 36 hours in this concentration, development proceeds with a varying percentage of partial apical death, but few total deaths. Such series give the usual degrees of differential inhibition, with more or less differential recovery in the apical region in 25 to 50 per cent, appearing as apical reconstitution in some of the partial forms. With a very short period of action, very high

LARVAL DEVELOPMENT IZ; THE SEA URCHIN 99

concentrations of KCK iiiay be used on blastula or gastrula stages, e.g., m;100, 2 to 2+ hours, m/200, 4 to 44 hours. With such concentrations and times total death occurs in 75 to 90 per cent, and a greater or less degree of apical partial death in the remainder. The partial basal forms which remain alive undergo more or less reconstitution, according as the living basal portion represents more or less of the apico-basal axis. The resulting forms show mostly the more extreme degrees of differential inhibition. A small percentage of narrow angle plutei (fig. 6) or forms with parallel or fused arms occur (figs. 82 to X T ) , also some forms with apical reconstitution (figs. 57 to Sl) , but 50 per cent or more of those which remain alive never develop beyond the spherical stage (figs. 19 to 21).

It is evident from these data that a wide range of concentrations of KCN may be used without altering the results very ureatly, except as regards the percentages of deaths and of dif- 7 fcrent forms. Within certain limits the chief difference is that., the higher the concentration, the larger the percentage of total deaths and partial deaths and so the smaller the percent- :tgc of living forms and the larger the percentage of partial forms n hich show no differential recorery or only apical reconstitution. Wherc the whole development occurs in KCK, differential acclimation is very slight, and occurs in very few individuals.

Ammonium 1, ydratc

In the case of NH,OH much higher concentrations than of KCPI: must be used to produce differential axial effects on development, but in concentrations high enough to produce such cff ecis, differential inhibitions with no appreciable differential :mlim&ion result. Eggs developing in NH,OH m/10000 from thc first cleavage produce practically normal forms, the only indication of differential inhibition being a slightly reduced oral lolw.

Eggs developing in NH,OH m/50@0 froni first, cleavage, show 25 to 50 per cent of total deaths and most of the remainder show partial apical death. SO to 90 per cent are spherical basal forms like figures 19 to 21, without reconstitution or differential

100 C . M. CHILD

acclimation. The remainder show various degrees of differential inhibition, the least inhibited being small plutei like figures 12 and 13. The various differential inhibitions are of the same types throughout as those produced by KCN, and no forms of any other sort appear in such cases.

Sodium hydrate

NaOH is much less toxic than NH,OH. Concentrations above m/500, acting continuously from the first cleavage, usually inhibit development or kill completely at the blastula stage. In m/500 from the first cleavage, a considerable percentage of total deaths occurs at or before the blastula, and most individuals show more or less apical death. The resulting forms show differential inhibition, usually of the more extreme types, and many remain spherical basal forms like figures 19 to 21.

In m/1000 from the first cleavage, inore or less apical partial death may occur in 10 to 40 per cent, but few or 110 total deaths. Of the resulting forms the majority show sonie degree of differential inhibition, but some are noriiial plutei, and some show :L slight degree of differential acclimation. -111 degrees of differential inhibition occur, and differential accliination appears only in the relatively large oral lobes and wide angles of divergence in some plutei (figs. 23, 24, 29).

In m,’2000 from the first cleavage, apical partial death may be almost entirely absent, w it may occur in 15 to 20 per cent of the blastulae and ranges from the death of a few apical cells to death of about the apical half. f e ~ v of the partial basal formh, iii which apical death has proceeded farthest remain spherical (figs. 19 to 21) or show some apical reconstitution, but the great majority of the embryos form plutei, almost all of which show the relatively large oral lobe characteristic of differential acclimation, and in 5 t o 10 per cent the angles of divergence are wider than normal (figs. 23, 24, 29)’.

In rn/5000 from the first cleavage, the effect on both rate of development and on form is slight. -lpic.;ti death does riot occur, and all forin plutei which are either ~iori~ial or possess a slightly larger oral lobe and slightly wider ::ngleh than nonnal.

LARVAL DEVELOPMENT IN THE SEA URCHIN 10 1

NaOH in sufficiently high concentration evidently retards or inhibits development and produces all degrees of differential inhibition in form. Differential acclimation occurs more rapidly and to a greater extent than in NH,OH, but still is not sufficient to produce the more extreme types of form characteristic of differential acclimation in alcohol and acids.

Alcohol

Development from the first cleavage in alcohol 4 per cent is greatly retarded, and apical death begins at or before the blastula stage, usually resulting in total death before or in the gastrula stage, but in a small percentage more or less of the basal region may remain alive in some lots and a few small spherical, partial forms like figures 20 and 21 may result.

In alcohol 3 per cent from the first cleavage, total death occurs in 20 to 50 per cent in the blastula or gastrula stage, and most of the others show more or less apical death. The resulting forms show the more extreme types of differential inhibition, and mostly remain without a skeleton.

Development is retarded in 2 per cent alcohol, but there is usually no death, and differential acclimation begins with apical outgrowth as early as the gastrula stage (figs. 25,26), and the final stages reached are forms like figures 40 to 48, without skeleton and with an elongated apical outgrowth representing the oral lobc, with a few like figures 49 to 53, which represent partial basal forms.

In 1.5 per cent alcohol development is less retarded and the degree of differential acclimation is greater. About half the resulting forms show inarked elongation and over-development of the oral lobe like figures 27, 28, 38, 39, and the remainder range through the forms of figures 40 to 48.

In 1 per cent alcohol plutei develop which range in form from slight degrees of differentid inhibition to slight degrees of differential acclimation, and in lower concentrations thereis usually no effect on form.

A few experiments on recovery after the temporary action of alcohol indicate the possibilities in this direction. Late cleavage stages in alcohol 4 per cent 11 hours, then washed and re-

102 C. M. CHILD

turned to water, advanced to the blastula stage during the alcohol period, but no movement occurred until returned to water. At the end of the alcohol period partial death, ranging from a few apical cells to the apical half had occurred in from 25 to 40 per cent. The resulting forms are all without skeleton and show differential inhibition of the more extreme degrees, with apical differential recovery indicated by apical outgrowth, as in figures 74 to 81, in 30 to 40 per cent. The remainder, mostly partial basal forms, are spherical with entodermal vesicle (figs. 19 to 21) and show no differential recovery.

Late cleavage stages in 3 per cent alcohol, 18 hours, advance to beginning of gastrulation during this period, but 10 to 20 per cent show a small amount of apical death. Resulting forms are from 30 to 40 per cent differentially inhibited plutei with more or less rudimentary skeleton like figures 7, 12, 13, and with no evidence of differential recovery; 30 to 40 per cent are askeletal rounded forms with apical outgrowth like figures 74 to 81; and the remainder are spherical with entodermal vesicle, mostly partial basal forms, without differential recovery. This case shows an interesting feature of the relation between differential inhibition and differential recovery. Those individuals which are least inhibited develop most rapidly and so attain an early pluteus stage before differential recovery occurs to any great extent. Consequently they show only the lesser degrees of differential inhibition. Those which are more susceptible are more retarded in their development, and even after return to water attain a less advanced stage of developmcnt, but, nevertheless, show a greater degree of differential recovery than the less susceptible, because renewed growth and development are limited to the apical region, while in the less susceptible individuals they occur more or less over the whole body. Finally, in the most susceptible individuals, morc or less of the apical region is killed, and differential recovery does not occur to any appreciable extent. In short, the forms characteristic of differential recovery can be produced only within certain limits of inhibitmion. If the degree of inhibition be not sufficient, or be too extreme, the degree of differential recovery is not sufficient to affect the form of the animal.


d c i d s

Development from the first cleavage in HC1 m / l O O O proceeds slowly, reaching the blastula stage in about 24 hours; with 50 per cent more or less of deaths, mostly total. During the next two or three days the mesenchyme cells enter the blastocoel, and after four or five days, gastrulation may begin. Even in this concentration the gastrulae begin to show apical outgrowths like figures 25, after six days in the solution, but do not usually develop further, and even if returned to water at this time they die in a day or two.

In HCl m/2000 from the first cleavage, a few total deaths occur and more or less of the apical region dies in 20 to 40 per cent at or before the blastula stage. From this stage on, differential acclimation begins, and the whole gastrulae are like figure 25, the preplutei, like figures 27 and 28, and the final stages range from the extreme types of differential acclimation shown in figures 31 to 36, through skeletal forms with apical outgrowth representing the oral lobe and with or without basal ciliated band, like figures 37, 60, 61, 64, and rounded forms with small apical outgrowth like figures 49, 51 to the lower limits of differential acclimation like figures 50, 52, 53, 78 to 81. In fact, practically every individual which lives through the gastrula stage, even as a partial basal form, shows at least some degree of differential acclimation.

In HCl m/5000 from the first cleavage there are usually no deaths, and apical outgrowth begins in the gastrula (fig. 25), and the resulting forms range from wide-angled plutei with large oral lobe (figs. 23, 24, 29 to 32), through normal plutei, to inhibited forms with apical acclimation (figs. 37, 60, 61, 64, 49, 51). The extreme types of differential acclimation like figures 33 to 36 do not usually appear in this concentration.

In HC1 m/10000 from the first cleavage, all develop into wide angled plutei with large oral lobe, about 50 per cent approach- ing normal, like figures 23 and 24, and the remainder ranging from these to forms like figures 29 to 32.

In HC1 m/50000 from the first cleavage, 50 per cent, more or less, are normal, the remainder slightly wide angled with large

104 C. M. CHILD

oral lobe, with forms like figure 23 as the maximum modification of form.

&Icetic acid was used only for comparison with HCl, and in two concentrations m/2000 and m/5000, acting continuously from the first cleavage. The forms produced were of the same type and range of variation as in the same concentrations of HC1.

With the acids, as with alcohol, it is evident that for the more extreme modifications of form by differential acclimation, a certain degree of differential inhibition is necessary as an antece- dent condition. The most extreme modifications of form by differential acclimation appear in acids because differential acclimation begins earlier and progresses more rapidly than in other agents, so that the relations of parts are widely altered before the skeleton develops.

GENERAL CONCLUSIONS

The experimental data leave no possibility of doubt concerning the effectiveness of the axial metabolic gradients as fundamental factors in the embryonic development of the sea urchin. The differences in susceptibility to inhibiting agent.s which are associated with the differences in metabolic rate at the different levels of the gradients, determine on the one hand the differential effects of direct inhibition and, on the other, those of differential acclimation and differential recovery.

Since there are no specific differences in the form-changes produced by the different agents used, there is no basis for the as- sumption of specific action in any case. All the facts indicate that the action of the various agents is essentially quantitative, so far as it concerns the processes of growth and development. It is very probable that the different agents do not all act in exactly the same way on the sea urchin protoplasm, but the point of present importance is that, however they act, whatever condition or reaction complex in the system they affect primarily, their general effect is a retardation or inhibition of the fundamental metabolic processes, which may be more or less completely reversed by acclimation or recovery. The’ changes in form result from the differences in effect on different regions;


and these in turn are dependent upon the differences in rate of reaction or in protoplasmic condition, permeability, aggregation, enzyme activity or whatever designations we prefer, associated with the differences in rate of reaction. The relation between susceptibility, and metabolic rate is a very general one, and is apparently independent, at least to a large extent, of the particular component reaction or condition of the protoplasmic reaction system which is directly affected in a particular case. It is dependent, rather, upon the fact that living protoplasm is a system and that no very great changes in any essential component of this system are possible without affecting the system as a whole.

It is evident that the alterations in form resulting from differential inhibition and from differential acclimation and recovery are very closely associated with changes in the relative rate and amount of growth a t different levels of an axis. The so-called normal form of the sea urchin, or of any other organisms, represents merely the usual relations of metabolism and growth between different parts.

Experimental data of many kinds and from many fields show- that where nutritive supply is limited, a region of high metabolic rate will grow or mainta.in itself more or less completely at the expense of a region of lower rate; i.e., some of the products of breakdown in the region of low rate go to the building up of new molecules in the region of high rate because the physico- chemical conditions determine a passage of these substances toward the region where they are being most rapidly transformed. In short, the region of higher rate of reaction robs the region of lower rate. Where the nutritive supply equals the demand in all parts, the region of higher rate of reaction shows, in general, a higher rate of growth than a region of lower rate, simply because it synthesises more molecules in a given time (Child, ’15 b, Chap. 11). General form of body and proportions of parts are, fundamentally, the expression of relations of this character. They represent, so to speak, the metabolic balance between regions of different rate of-reaction under a particular complex of conditions. The ‘normal’ form is merely one par-

106 C. M. CHILD

ticular case, or, more correctly speaking, a certain limited range of variation in metabolic relations, and it is normal, merely because, under the usual conditions, this range of variation is not exceeded.

Not only differential growth, but local differentiation, may occur at different points of a metabolic gradient. Some of the evidence in support of the view, that local differentiation results, in the final analysis, from the differences in metabolic condition which arise at different points of a gradient, in consequence of the differences in rate of reaction, has been considered elsewhere (Child, ’15 c, pp. 127-169, 183-188). If this view be correct, ‘normal’ localization of differences in development is like general form and proportion merely, one particular case or a certain limited range of cases representing a certain limited range, the ‘normal’ range of variation in the essential metabolic conditions.

The experimental methods used in the present paper serve merely to alter the differences in metabolic rate betweendiffer- ent parts and so to alter the resulting balance and therefore the spatial order as expressed in growth, form, and differentiation. In differential inhibition the slope of the metabolic gradient is decreased, the gradient is more or less completely leveled down, because the regions of high rate of reaction are more susceptible to the action of the inhibiting agent and so undergo a greater decrease in rate, than the regions of lower rate. In consequence of this decrease in slope of the metabolic gradient, the differences in metabolic rate between different points of the gradient, and therefore the differences in rate and amount of growth, become less than in the normal animal, and the relative size and proportions of parts along any axis are altered, those parts which represent regions of high rate of reaction becoming relatively smaller and those which represent regions of low rate, relatively larger. As this decrease in slope of the metabolic gradient progresses the gradient becomes less effective as a factor in differentiation, and local morphological features along its course may become less and less marked and finally disappear, or more correctly, fail tro appear.


In the various degrees of differential inhibition described, the progressive obliteration of metabolic gradients, i.e., of axes appears. Bilaterality may be almost completely obliterated, while longitudinal and apico-basal gradients still remain effective (figs. 86,87) ; and in other cases both bilaterality and the longitudinal axis are practically obliterated and the apico-basal axis remains as the chief determining factor in growth and differentiation. This condition is most evident where differential acclimation or recovery occurs in the apical region, but there is little or no indication of other axes in the larva. Figures 40 to 53 and 57 to 81, show various stages in this axial obliteration. The limit in this direction is the obliteration of all axes, including the apico-basal axis. This limit is approached or perhaps attained in some cases (figs. 19 to 21). Under these conditions definite progressive development and localized differentiation cease, although life may continue until ended by starvation.

In differential acclimation and differential recovery the changes in metabolic relations between different points of a metabolic gracient are in the opposite direction from those in differential inhibition. Since, with the method employed, differential acclimation and recovery are possible only after differential inhibition, it may happen that metabolic gradients are so far obliterated by differential inhibition that differential acclimation or recovery is impossible, but where the gradient is not obliterated to this extent, differential acclimation and recovery consist in an intensification, a steepening of its slope, beginning at the high end. The metabolic differences between different points of the gradients are increased, and the form and proportions of the larva and the positions of localized differentiations show changes in the opposite direction from those characteristic of differential inhibition. The apical region develops at the expense of the basal, the anterior at the expense of the posterior and the median at the expense of the lateral.

The differences between the forms resulting from differential inhibition and differential acclimation show to what extent the general form, proportions and localization of parts can be altered and controlled in this way. It is possible to transform the

108 C. M. CHILD

larvae in one direction into apico-basally elongated forms, with slight apical development, with little or no trace of longitudinal axis or bilateral symmetry, or, in the extreme case, into a spherical form, with little or no trace of any axis, and in the other into a transversely flattened form with very great apical, anterior and median over-development, and the localization of parts differs correspondingly in the two types. The direction of the skeletal rods may be shifted through ninety degrees, or the skeleton may be completely inhibited or very greatly over-developed. I t would be difficult, I think, to find a more complete demonstration of the effectiveness of the metabolic gradients in development than these form-changes in the sea urchin. We are able to modify, to control, and to predict the changes in form and relations of parts which occur.

The fact that differential effects on development result from the action of inhibiting agents, not only upon the various stages of development themselves, but upon the unfertilized egg .is also important. It may appear, at first glance, that this fact constitutes a demonstration of the actual existence of the definitive axial metabolic gradients in the unfertilized egg, but this is not necessarily the case, as a moment’s consideration will show. Assuming that no metabolic gradients are present in the unfertilized egg, the effect of the inhibiting agent must be the same on all parts of the protoplasm or of its limiting surfaces. If this effect persist after fertilization, the protoplasm is less capable of stimulation and therefore, if the metabolic gradients arise de novo during or after fertilization, it is conceivable that differences in metabolic rate in such gradients may be less than in normal eggs. If, on the other hand, recovery from the inhibiting effect is occurring at the time the metabolic gradients arise, the differential acceleration of metabolic rate associated with the establishment of a metabolic gradient may itself determine a differential recovery, since the rate and degree of recovery varies with metabolic rate. In short, a general inhibition preceding the establishment of the metabolic gradients, if its effect persist during the period of establishment, may determine that the metabolic differential, the metabolic slope of


such gradients, when they do arhe, shall differ from that produced in the normal egg by the same factors. The demonstration of the existence of metabolic gradients in the unfertilized egg by this method is then not complete. The evidence from death gradients is also unsatisfactory on this point, because, with the low metabolic rate in the unfertilized egg, differences in rate are at least slight (Child, 16 c). According to Boveri ('01 a, '01 b) the apico-basal axis of embryo and larva coincides with the axis of the growing oocyte in Strongylocentrotus lividus, but Garbowski ('05) has shown that such coincidence does not occur in all cases, and, according to Wilson and Mathews ('95), the embryonic axis in Toxopneustes may form any angle with the axis determined by the position of the polar bodies and of the nucleus after maturation, while in Asterias these two axes coincide.

These various observations afford no adequate basis for general conclusions. If they are all correct we must conclude that in echinoderms the apico-basal axis of the embryo may coincide with that of the growing oocyte, or may be determined de m u 0

by factors acting on later stages. If the original axis of the growing oocyte is a metabolic gradient, it is evidently not very strongly marked or permanently fixed in the protoplasm, and other factors acting differentially on the egg (Child, '15 b, Chaps. 11, V.) may determine a new effective gradient, as is apparently the case in Asterias (Wilson and Mathews, '95; Child, '15 a), and as Garbowski's observations indicate in some cases in Strongylocentrotus (Garbowski, '05). If any metabolic gradient is present in the unfertilized egg of Arbacia it is apparently slight (Child, '16 c), and it seems probable that the differential conditions associated with fertilization, or perhaps, in some cases, the differential action of other external factors might determine a new effective gradient. In general, the observations, as far as they go, suggest that the apico-basal embryonic axis coincides with the axis of the growing oocyte, except where the differential action of other factors is sufficient to determine a new effective gradient, and this is very probably true for many other animal eggs.

110 C. Y. CHILD

As regards the longitudinal axis and bilaterality in Arbacia, the fact that they do not become visibly effective factors in development until later stages, and also the fact that they can be experimentally almost or quite obliterated while the apico-basal gradient still persists, suggest that they are less marked as metabolic gradients and perhaps determined later than the apico- basal axis. Conceivably they may arise in connection with fertilization through the differential effect on the egg protoplasm of the positions or paths of the pronuclei and the direction of the spindle axis. But at present no final conclusion is possible, and, as pointed out above, the differential effects on later development of the action of inhibiting agents on unfertilized eggs do not constitute a demonstration of the existence of the definitive axial gradients in the unfertilized eggs.

Attention has already been called to the fact that some of the differentially inhibited forms resemble, in certain respects, the more primitive types of echinoderm larvae as regards the complete closure of the blastopore, the apico-basally elongated, more or less cylindrical, conical or ovoid body without strongly marked antero-posterior axis or bilaterality, and the development of the basal portion of the ciliated band as a more or less complete basal ring (p. 79). It may also be noted that the wide-angled forms resulting from differential acclimation show some resemblances in form to certain ophiurid plutei. These resemblances suggest that the condition of the axial metabolic gradients in the experimentally produced forms of Arbacia and the normal forms which they resemble, is somewhat similar. The fact that certain degrees of obliteration of the axial gradients by differential inhibition produce an approach to more primitive larval types, is particularly interesting as suggesting that the evolution of the pluteus has consisted to some extent in an evolution of the axial gradients, or, more correctly speaking, of the protoplasmic conditions which determine the establishment, metabolic slope and physiological effectiveness of such gradients. The pluteus, in short, shows a higher degree of axiation than the crinoid larva, i.e., the axes appear more distinctly in form and differentiation of the body, and there is

LL4RVAL DEVELOPMENT I N THE SEA URCHIN 111

reason to believe that certain of the minor metabolic gradients in the pluteus are not present in the crinoid larva; certainly they are not effective factors in larval development.

The modifications of form produced by differential inhibition and differential acclimation and recovery, demonstrate the real- ity of the metabolic gradients as effective dynamic factors in the development of Arbacia. The analysis of these modifications indicates that the apical region is the region of highest rate of reaction in the organism; or in other words, that the region of highest rate of reaction becomes the apical region. From the apical region the rate of reaction decreases in all directions, but less rapidly on that side which becomes anterior than on that which becomes posterior, and apparently less rapidly along the median region than laterally. The development of the skeleton of course modifies these simple relations and determines new localized regions of growth and so new local gradients.

The method of demonstrating the axial metabolic gradients by the death gradients in lethal concentrations of inhibiting agents (Child, '13 b, '14 a, '15 a, '15 c, Chap. 111, '16 a) is much less delicate than the method of modifying development by differential inhibition and differential acclimation or recovery, but the conclusions reached by means of the cruder method, so far as they go, are confirmed, and further conclusions made possible by the more delicate method.

A few data not yet published on differential inhibition in the larvae of the starfish and of certain polychete annelids, indicate that the metabolic relations can be altered along at least the apico-basal gradient in the same way as in Arbacia. Moreover, in the reoonstitution of isolated pieces of Planaria, differential inhibition and different.ia1 acclimation and recovery in the longitudinal and transverse axial gradients can be brought about in the same way as in the sea urchin larva, and the resulting modifications of form are of the same character. In differential inhibition anterior (apical) regions are most inhibited, posterior least, and organs normally bilateral, such as the eyes and the cephalic lobes, approach the median line and, in the more extreme degrees of differential inhibition, become median (Child,

112 C. M. CHILD

'12, '16 b), while in differential acclimation and recovery the changes are opposite in direction. The fact that axial relations in these widely different organisms can be experimentally altered and controlled in similar ways through the differential effect of inhibiting agents indicates very clearly the fundamental identity of the physiological axes as metabolic gradients. In the light of these facts, the further demonstration of the existence of axial metabolic gradients in algae among plants (Child, '16 a) and in protozoa, coelenterates, flatworms, echinoderms, annelids, fishes, amphibia and birds among animals and their correspondence with developmental gradients of other kinds are sufficient to establish the general and fundamental significance of such gradients in the development and differentiation of organisms . I

The modifications of form, localization, and differentiation by differential inhibition, acclimation and recovery in the sea urchin larvae and in the reconstitution of pieces of Planaria afford a basis for the interpretation of many other cases of experimental teratogeny and of various teratological forms observed in nature. The cases of cyclopia in fishes produced experimentally by Stock- ard and others are simply differential inhibitions like those produced in the sea urchin and Planaria. Median regions are more inhibited than lateral, and lateral organs approach the median line or become median. Differential inhibition in the antero- posterior axis in the early stages of development may produce various gradations from the normal form to acephaly. In seg- mented animals, where a region of high metabolic rate arises secondarily at the posterior end of the primary gradient and becomes a growing region from which new segments develop, a secondary gradient arises, with its region of highest rate in the posterior growing region, and differential inhibition along this gradient may produce certain characteristic teratological forms.

In short, the examination of teratological forms which are not the direct result of some factor acting locally, but are produced by agents or conditions which affect the body as a whole,

IChild, '12, '13 b, '14a, '15a, ' 1 5 ~ ' 'lGa, '16 b, 'lGc, Hyman, '16. A considerable part of these data is still unpublished.


indicates very clearly that differential susceptibility along metabolic gradients plays a very important part in their production. And as I have shown for the sea urchin, the same agent or condition may not only produce different degrees of modification in a particular direction, according to concentration, period of action, etc., but may produce modifications in opposite directions, the one direction representing differential inhibition, the other differential acclimation or differential recovery. On the other hand, very different agents and conditions may produce similar modifications of form, because the general effect upon metabolism is not specific, but quantitative. The facts indicate that the fundamental factors concerned in these modifications are alterations in one way or another of the rates of reaction and so of the metabolic relations of parts.

-4s a matter of fact, experimental teratogeny affords the most conclusive evidence of any field of investigat.ion for the fundamental significance of metabolic gradients in orderly development and differentiation, for the modification and control of development by modification and control of the metabolic rates and relations in these gradients enables us to determine and test their effectiveness. Experimental teratogeny has suffered from the failure to find a general foundation for experimental procedure, analysis and interpretation, but from the view point which we attain with the aid of the conceptions of metabolic gradients and differential susceptibility, a wide field lies before us ready for logical experimentation, analysis and synthesis.

SUMMARY

1. If the directions or axes to which the order apparent in development is related are fundamentally gradients in rate of metabolic reaction, it must be possible to alter the developmental order as expressed in form, proportion and localization of parts by altering the relations of rate of reaction along these axes or gradients.

2. The general relation between susceptibility to a great variety of agents and conditions which retard or inhibit metabolic reaction in one way or anot'her and rate of metabolic reaction,

J O C l I S A L OF NORPHOLOQY, VOL. 28. N O . 1

114 C. M. CHILD

affords a means of altering the relations of metabolic rate along such gradients. The relation between susceptibility and metabolic rate is briefly as follows: in concentrations or degrees which kill without permitting acclimation, susceptibility varies directly with metabolic rate. In low concentrations, where acclimation occurs, the rate and degree of acclimation vary directly with metabolic rate. Where the inhibiting agent acts only temporarily the rate and degree of recovery vary directly with metabolic rate.

3. ,Acclimation to, and recovery from the action of the inhibiting agents used in these experiments consists in the attainment of a higher rate of reaction, either in the presence of the agent or after its action. 4. Potassium cyanide, ethyl alcohol, amnionium hydrate, so-

dium hydrate, hydrochloric and acetic acid in all concentrations above a certain minimum retard or inhibit development of the sea urchin, but with sufficiently low concentrations, a greater or less degree of acclimation may occur in the presence of the agent, or a greater or less degree of recovery after temporary action.

5 . -4 graded difference in susceptibility to these agents exists dong the axes of developmental stages of the larval sea urchin body. This difference in susceptibility appears either as differential inhibition, differential acclimation, or differential recovery dong the axes, according to the concentration of the agent used and the period of action.

6. . These differential effects along the axes bring about characteristic changes in form, proportions and localization of parts of the larval body, which can be experimentally controlled and predicted t o a very considerable degree.

7. The larval forms resulting from differential inhibition show changes in form, proportions and differentiation in a certain direction from the normal, the degree of change corresponding to the degree of differential inhibition. The chief changes are decrease in size of oral lobe, which represents the apical region, decrease to zero in the angle of divergence between arms, approach of lateral parts toward the median line and in more extreme degrees fusion in the median line, the progressive oblit-

LARVAL DEVELOPMEKT IN THE SEA URCHIS 115

eration of antero-posterior and medio-lateral differences and finally of apico-basal differences.

8. The changes in differential acclimation and differential recovery are in the opposite direction. They consist in increase in size and over-development of the oral lobe, increase to 180" in the angle of divergence between the arms, and over-development of ant,erior and median as compared with posterior and lateral regions.

9. Where the effect of differential inhibition persists after general recovery, a great over-development of the skeleton may occur, since the mesenchyme cells are relatively less inhibited than most, if not all other parts.

10. Forms produced by the more extreme degrees of differential inhibition resemble, in certain respects the forms of the more primitive echinoderm groups, and the wide-angled plutei resulting from differential acclimation resemble larvae of ophiu- ride. These facts suggest that in the evolution of the pluteus larva from the primitive larval form, changes in the metabolic relations along the axial metabolic gradients and perhaps the establishrncnt of conditions which determine new gradients, have played an important part.

11. A11 the experimental data indicate that the spatial orders in the larval development of the sea urchin are fundamentally gradients in rate of general metabolic reaction, with highest rates of reaction determining apical, anterior and median regions.

12. The different agents used differ in degree in their inhibiting action, but, there is no evidence in the changes of form produced of any specificity of action. The order of decreasing effectiveness in inhibiting development is as follows : cyanide, ammonium hydrate, sodium hydrate, ethyl alcohol, acids. This order is also the order of increasing capacity of the sea urchin to become acclimated to, or to recover from the effects of these agents.

13. The relation between susceptibility and metabolic rate affords a method for analytic teratogenic investigation and a basis for the interpretation of many cases of experimental teratogeny already recorded and also of many teratological forms observed in nature.

116 C. M. CHILD

LITERATURE CITED

BO~ERI, T. 1901 a Uber die Polaritat des Seeigeleies. Verh. phys.-med. GeH. Wurzburg, N. F., Bd. 34. 1901 b Polaritat von Ovocyte, Ei und Larve des Strongylocentrotus lividus Zool. Jahrbucher: Abt. f. Anat. und. Ont., Bd. 14.

1912 Studies on the dynamics of morphogenesis and inheritance in experimental reproduction. IV. Certain dynamic factors in the regulation of Planaria dorotocephala in relation to the axial gradient. Jour. Exp. Zool. vol. 13. 1913 a Studies, etc. V. The relation between resistance to depress- ing agents and rate of reaction in Planaria dorotoccphala and its value as a method of investigation. Jour. Exp. Zoiil. vol. 14. 1913 b Studies etc. VI. The nature of the axial gradients in Pla- naria and their relation to antero-posterior dominance, polarity and symmetry. 1914 a The axial gradient in ciliate infusoria. 1914 b Starvation, rejuvenescence and acclimation in planaria dorotocephala. Arch. f . Entwickelungsmech., Bd. 38. 1915a Axial gradients in the early development of th- starfish. h e r . Journ. Physiol., vol. 37. 1915 b Senescence and rejuvenescence. Chicago. 1915 c Individuality in organisms. Chicago. 1916 a Axial susceptibility gradients in Algac. Bot. Gazette, 4'2. 1616 b Studies on the dynamics, etc. IX. The control of head-form and head-frequency in Planaria by means of potassium cyanide. Jour. Exp. Zobl. vol. 21. 1916 c Bxial susceptibility gradients in the rarly development of the sea urchin.

GARE~OWSKI, T. 1605 Uber die Polaritat des Sceigeleics. Bull. Acad. Sci. Cracovie; C1. Sci. math. e t nat., Oct.

HYMAN, L. H. 1916 An analvsis of the process of regeneration in certain micro- drilous oligochetes.

WILSON, E. B. and A. P. MATHEWS 1895 hlaturation, frrtilization and polarity in the echinoderm egg.

CHILD, C. M.

Arch. f . Entwickelungsmech., Bd. 37. Biol. Bull., vol. 26.

Biol. Bull., vol. 30.

Jour. Exp. Zool., vol. 20.

Jour. Morph., vol. 10.

PLATES

117

EXPLANATION 01.' P L . i m s

All figures arc drawn from living individrials; nntl form, proportions, arid axial relations arc reproduccd as exactly as possil)?c, but they are otherwise semi-diagrammatic in t.hat, det,ails not esscntial to thc purpose of the pnpcr are omitkd. Tlir ciliated baiid on ihe nntcrior end and arms of the pluteus is drawn only in figures .i 21 and h' to indicate its nornial rclations and in figures 37 t o 43, 47, 43, 57 t o 72, i 4 to 57, wherc it,s devclopmcnt is modified. In olhcr figures of plut,ei (figs. 6, 7, 23, 24, 29 to 36, 82 to SO), where its dcvelopmcnt shows essentially thc usual relation to that of the antcrior end, it is omittcd. The niescnchyme cells are omittcd in a11 cases, and thc fenestration of thc arm rods ?nd thc posterior skclctal structure is nicrcly rliagrammaticnlly indicatcd. In figurcs of blastulac and gnstrulae, all park are drawn in tloul~lc contour t o in&- cate the thickness of the body layers. In figurcs of stagcs intermediate bctwecn gastrula and pluleus singlc contours are used, exccpt for the thick-wnllcd storn- ach-intestine, and in figurcs of pluteus stages sin& contours arc used throiighout.

Figures lettered A , B, C with thc same numbcr arc, respcctivcly, bnsal (anal) lateral and anterior vicws of the sanic individunl except in thc case of figure 74 where B is a lateral and H nn anterior view.

P L x m 1

EXPLANATION OF FIGURI3S

Normal development 1 to ,j Elongated blastula to fully drvclopctl plutcut..

118

LAl lVAL UE\ PX,OI’AlEXT IN THE SEA URCHIK C. AI. CHILD

PLATE 5

4

5B

119

PLATE 2

EXPLANATION OF FIGURES

Differential inhibition. 6 A B Narrow-angled pluteus with somewhat inhibited oral lobe. 7 A B Final stage with greater degree of differential inhibition. 8, 9 Differentially inhibited blastula aiid gastrula. 10, 11 Exogastrulation. 12, 13 Final stages of more extreme differential inhibitions showing decrease

14 Anenteric pluteus, probably from an exogastrula. 15 Askeletal form. 16 Spherical anaxiate form. 17, 18 Partial basal gastrulae. 19 to 21 22 Degeneration of enteron in spherical forni.

of angle of divergence of skeletal rods.

Final stages of small basal forms.

120

LARVAL DEVELOPMENT IN THE SEA URCHIN C. H. CHILD

PLATE 2

15 21

26 22

121

PLATE 3 EXPLANATION OF FIGURER

Differential acclimation 23, 24 Wide-angled plutei. 25 Differential apical acclimation in gastrula. 26 Apical acclimation with basal irregularity in alcohol gastrula 27, 28 Aplcal acclimation in preplutei.

122

l..\P.V.iI, DEVELOPMEKT IN THE SEA URCHIN C. 11. CRlLD

PLATE 3

PLATE 4

EXPLANATION OF FIQURES

Difierential acclimation 29 to 33 Cases of differential acclimation showing over-development of

apical and median anterior regions as compared with basal, lateral and posterior regions.

124

PLATE 4 LARVAL DEVELOPMENT IN THE SEA URCHIN C M. CHILD

31A 3oB

G@ hB & 33B

32A

33 c 125

PLATE 5


Differential acclimation 34 to 36 Further cases of over-development of apical and median anterior

37 Differential acclimation after greater degree of inhibition, showing modi-

38 A larva showing apical acclimation and basal inhibition. 39 Apical acclimation arid basal inhibition.

regions as compared with basal, lateral and posterior.

fication in course of basal portion of ciliated band.

126

LARVAL DEVELOPMENT IN THE SEA URCHIX C. M. CHILD

PLATE 5

35A

35c

37A

38B 127

37 c

39

PLATE 6 EXPIANATION OF FIGuaEB

Differential acclimation and differential recovery 40 to 48 Cases of apical acclimation after the more extreme degrees of dif-

ferential inhibition, in which the antero-posterior axis and bilateral symmetry have been more or less completely obliterated.

49 to 53 Cases illustrating the lower limits of differential acclimation after extreme differential inhibition which has obliterated all axes except the apico- basal.

54 to 56 Differential recovery in early and late grastrulae and early pluteus. 57 to 62 Cases of differential recovery after a considerable degree of dif-

ferential inhibition, showing rudimentary oral lobe as an apical outgrowth, and a basal portion of ciliated band; antero-posterior axis and bilaterality more or less completely obliterated: figure 62 from an exogastrula.

63, 64 Cases of differential recovery with rudimentary oral lobe, but without basal portion of ciliated band.

65 to 67 Cases of differential recovery in which oral lobe does not develop, mouth is apical and basal portion of ciliated band is present; in figure 67 the enteron has separated from the blastopore region and a lateral anus has formed.

IAHVAI, DEVELOPMENT I N THI: SF: 4 UliCII IN C Y. CHILD

I'JA'l'E 6

45 43 44

8 49 50 47 48 b 53 5 4

52 55 56

58 60 61 57 59

I’LATE 7

EXPLANATIOS OF FIGURES

DiEerential recovery and differential inhibition with general recovery 68 to 73 Further cases of differential recovery, showing various differcnces

74 to 77 Cases in which differential recovery is limited to the developnirnt

T8 to 81 The lower limits of differential k o v e r y . 82 to 84 Persistent differential inhibitions after gencriil recovery showing

under-development of apical and mcdian :interior regions :ts compsrcd with basal lateral and posterior.

in detail; figure 71 from an exogastrula.

of a rudimentary oral lobe; figure 77 a partial basal form.

130

PLATE 8


Differential inhibition with general recovery 85 to 87 More extreme dcgrees of persistent differential inhibitiori with

88 to 90 Over-development of skeleton in cases of slight degrees of persist- general recovery.

ent differential inhibition with general recovery.

132

LARVAL DEVELOPMENT IN THE SEA URCHIS PLAYK x C. M. CHILD

86 A

88

133

experimental control and modification of larval development in the sea urchin in relation to the...

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